Estradiol metabolites as isoform-specific inhibitors of human glutathione S-transferases

Abstract

Numerous studies have suggested that the lifetime dose of unopposed estrogen is a significant risk factor for breast and uterine cancer. Estradiol (E₂) plays a putative role as a tumor promoter through interaction with estrogen receptors but can also be metabolized to redox active and/or mutagenic semiquinones and quinones. Similarly, equine estrogens (components of certain hormone replacement therapy preparations) are converted to quinone metabolites. The use of hormone replacement therapy has also been associated with increased breast and endometrial cancer risk. Recently, metabolites of certain equine estrogens have been shown to inhibit human glutathione S-transferases (hGSTs). Since E₂ and equine estrogens share similarities in other biological interactions, we have investigated the inhibitory capacity of endogenously formed E₂ metabolites toward various hGSTs. The quinone metabolite of 2-hydroxy-17-β-estradiol (2-OH-E₂) was synthesized, and inhibition of hGST-mediated biotransformation of model substrates was assessed. Inhibition of purified recombinant hGSTM1-1 and hGSTA1-1 occurred in a concentration-dependent manner with IC₅₀-values of approximately 250 and 350 nM, respectively. hGSTs M2-2, P1-1 and T1-1 were significantly less sensitive to inhibition. Specific glutathione-conjugates of the estrogen quinone also potently inhibited hGSTM1-1 and hGSTA1-1. Mass spectrometry data indicate that the inhibition was not mediated via covalent adduction. Although we have demonstrated hGST inhibition via E₂ metabolites, our findings indicate that the isoform specificity and potency of GST inhibition by endogenous E₂ metabolites is different than that of equine estrogen metabolites.

Introduction

The glutathione S-transferases (GSTs) are a multigene family of biotransformation enzymes capable of catalyzing glutathione (GSH) conjugation to exogenous and endogenous electrophilic species (for review see [1]). The GSTs have also been implicated in modulation of stress-activated protein kinase activity [2], [3], [4], protection against lipid peroxidation [5], [6] and transport of cellular molecules such as steroids [7]. The mammalian GST family is divided into at least eight classes (alpha (A), kappa (K), mu (M), omega (O), pi (P), sigma (S), theta (T), and zeta (Z)) with multiple subfamilies per class [8], [9], [10], [11], [12], [13], [14]. Individual GST isoforms demonstrate unique, though often overlapping, substrate specificity and range of cellular functions [1].

Estradiol (E₂) is a major circulating form of estrogen in humans and has been associated with the development of breast and endometrial cancers (for review see [15], [16]). E₂ is thought to increase cell proliferation via estrogen receptor-mediated cell signaling thereby acting as a tumor promoter; however, oxidative metabolites of E₂ are also considered to be mutagens (for review see [17]). To combat symptoms of menopause associated in part with declining endogenous estrogen levels, hormone replacement therapy is often prescribed. However, this treatment is similarly associated with increased risk of estrogen-related cancers [18], [19].

Equine estrogens (i.e. equilin and equilenin) are major components of certain estrogen replacement therapies such as Premarin (Wyeth-Ayrest) [20]. E₂, equilin and equilenin are metabolized to catechol estrogens and further to quinones via autooxidation (in the cases of equilin and equilenin) or via cytochrome P450 and/or peroxidase mediated catalysis (in the case of E₂) (Fig. 1) [20], [21], [22], [23], [24], [25], [26]. Upon quinone formation, the molecules may spontaneously react with glutathione [27], [28].

Metabolism is thought to modulate estrogen toxicity. For instance, quinone metabolites are capable of DNA adduction and free radical generation [29], [30], and in vivo research indicates that 4-hydroxylation is an activation reaction [31]. In the Syrian hamster and CD1 mouse models, 1,3,5(10)-estratriene-3,4,17β-triol (4-OH-E₂) treatment results in higher tumor incidence than 1,3,5(10)-estratriene-2,3,17β-triol (2-OH-E₂) treatment [31], [32]. Furthermore, in target organs sensitive to estrogen-induced cancer, 4-hydroxylation of estradiol predominates over 2-hydroxylation [33], and as opposed to the stable DNA adducts associated with E₂2,3-Q, E₂3,4-Q forms depurinating adducts with DNA [34], [35].

It has recently been established that metabolites of equine estrogens, equilenin and equilin, specifically and irreversibly inhibit human GSTs (hGSTs) [36], [37], [38]. Inhibition of hGSTM1-1 and hGSTP1-1 occurred at concentrations in the micromolar range via irreversible adduction to cysteine residues and oxidative damage [36], [37]. However, whether hGST inhibition by equine estrogens is unique in potency and isoform specificity, or whether endogenously formed estradiol metabolites are capable of similar inhibitory effects on human GSTs is unknown. Therefore, we sought to determine whether reactive metabolites of E₂ have interactions with GST similar in isoform specificity and potency to exogenously administered equine estrogens. Our aim was to characterize GST inhibition by endogenous quinone and glutathione conjugate metabolites of E₂. We have identified isoform-specific inhibition of hGSTs with IC₅₀values in the nanomolar range.